Genetic separation of Brca1 functions reveal mutation-dependent Polθ vulnerabilities

Homologous recombination (HR)-deficiency induces a dependency on DNA polymerase theta (Polθ/Polq)-mediated end joining, and Polθ inhibitors (Polθi) are in development for cancer therapy. BRCA1 and BRCA2 deficient cells are thought to be synthetic lethal with Polθ, but whether distinct HR gene mutations give rise to equivalent Polθ-dependence, and the events that drive lethality, are unclear. In this study, we utilized mouse models with separate Brca1 functional defects to mechanistically define Brca1-Polθ synthetic lethality. Surprisingly, homozygous Brca1 mutant, Polq−/− cells were viable, but grew slowly and had chromosomal instability. Brca1 mutant cells proficient in DNA end resection were significantly more dependent on Polθ for viability; here, treatment with Polθi elevated RPA foci, which persisted through mitosis. In an isogenic system, BRCA1 null cells were defective, but PALB2 and BRCA2 mutant cells exhibited active resection, and consequently stronger sensitivity to Polθi. Thus, DNA end resection is a critical determinant of Polθi sensitivity in HR-deficient cells, and should be considered when selecting patients for clinical studies.

loss of heterozygosity, at the cost of deletion or addition of a few nucleotides at repair sites 16,17 .TMEJ signatures at DSB sites can be distinguished by the presence of deletions formed through the annealing of 2-6 bp flanking microhomology, and/or templated insertions at DSB sites.Such sequences are associated with Signature 3, which is frequently observed in HR-defective cancers 12,[18][19][20] .Indeed, seminal studies revealed a synthetic lethal relationship between Polθ and HR repair encoding genes [21][22][23][24] .
BRCA1 mutation-containing cancers demonstrate varying responses to PARP inhibitor (PARPi) therapy, in part due to differences in the severity of HR malfunction 25 .The presence of BRCA1 hypomorphic proteins lacking various functional domains [26][27][28][29] , as well as mutations in DNA end resection genes such as 53BP1, can promote HR and PARPi resistance 30,31 .In contrast to PARPi, mutations in 53BP1 and other DNA end resection regulatory proteins increase cellular sensitivity to Polθ inhibitors (Polθi) 11,32,33 .However, the impact of distinct BRCA1 mutations and functional defects on cellular and organismal Polθ dependency is unknown.Moreover, several BRCA1 and BRCA2 mutant cancer cell lines show limited sensitivity to pharmacologic inhibition of Polθ activity 32,34 .Biomarkers that predict Polθi sensitivity within BRCA1/2 mutation carriers would be important for identifying optimal patient populations and informing clinical trials.
Here, we set out to assess Polθ dependency in mice and derived cell lines with mutations that disrupt distinct Brca1 functions, specifically in DNA end resection versus Palb2-Brca2-Rad51 recruitment.Furthermore, we characterized the effects of mutations in conjunction with loss of 53bp1, revealing new insights into cellular and organismal Polθ dependency, with relevance for the clinical application of Polθi.

Genetic context governs Polθ-dependent viability
To test if distinct Brca1 functional defects confer similar Polθ dependencies, we used previously established murine Brca1 Δ11 and Brca1 CC mutation-containing alleles.The Brca1 Δ11 allele produces a truncated protein that is defective for counteracting 53bp1, resulting in a failure to initiate DNA end resection 1 .In contrast, the Brca1 CC protein contains a coiled-coil (CC) domain deletion 35 , which has little impact on DNA end resection but is highly disruptive for Palb2 binding, resulting in failed Palb2-Brca2-Rad51 loading at DSBs (Fig. 1A).Homozygous Brca1 Δ11/Δ11 and Brca1 CC/CC MEFs with 53bp1 +/+ or 53bp1 −/− genotypes were readily ascertained from crosses of heterozygous transgenic mice, and were subsequently transformed with SV40.Each genotype demonstrated the expected Brca1 and 53bp1 protein expression patterns (Fig. 1B).
To gain insight into the makeup of marker chromosomes, we performed mFISH analyses on Brca1 CC/CC , Polq +/+ and Brca1 CC/CC , Polq −/− cells.Consistent with the G-banding studies, Polq −/− karyotypes showed many more derivative chromosomes (structurally rearranged chromosomes generated by two or more chromosomes, e.g., the unbalanced product of a translocation) than Polq +/+ karyotypes (Fig. 2D), with most of these rearranged chromosomes appearing to be clonal (Supplementary Fig. 2d, Supplementary Data 2, 3).Strikingly, several Brca1 CC/CC , Polq −/− marker chromosomes consisted of segments derived from up to 3 or even 5 distinct chromosomes (Fig. 2D, Supplementary Data 2-3).Therefore, Polθ loss exacerbates chromosomal breakage and rejoining in Brca1 mutant cells, the consequences of which, likely manifest in slowed cell division.

Brca1 mutations impact Polθi sensitivity
Small molecule Polθi is under development for the treatment of BRCA1/2 mutant cancers, and genotype-conferred sensitivity profiles could be useful for identifying optimal patient groups.To assess responses to pharmacological targeting of Polθ, colony formation assays were carried out with the small molecule Polθi ART558 32 .Cell line sensitivity profiles were compared to the PARPi rucaparib.

Brca1 mutations have distinct DSB outcomes
The severity of phenotypes observed with both genetic Polq KO and ART558 sensitivity was variable, with Brca1 Δ11/Δ11 cells showing mild, and Brca1 CC/CC cells a more severe growth slowing (Fig. 2A), or reduction in colony formation (Fig. 3B), respectively.However, when combined with 53bp1 deficiency, both Brca1 Δ11/Δ11 and Brca1 CC/CC cell lines demonstrated genetic synthetic lethality with Polq KO and increased sensitivity to ART558, despite also showing increased resistance to PARPi.We next aimed to gain insight into the molecular drivers of genotype-conferred Polθ dependency.Of note, we did not observe significant differences in Polq mRNA expression between genotypes (Supplementary Fig. 3a).
We next explored DNA repair events typically associated with HR and TMEJ.DNA end resection stimulates both HR and TMEJ 11 , and we confirmed previous results showing that Brca1 CC/CC are proficient, and Brca1 Δ11/Δ11 cells defective for resection measured by END-seq 35 , which directly measures ssDNA lengths 37 .As expected, 53bp1 KO increased resection lengths across all genotypes (Fig. 4C).RPA32 γ-irradiationinduced foci (IRIF) was used as another indirect measurement of resection and generally reflected END-seq trends (Fig. 4D).Together, these results re-enforce that RPA32 foci are reflective of DNA end resection products, i.e., ssDNA overhangs.We confirmed low levels of Rad51 IRIF in Brca1 mutant, 53bp1 +/+ cells.Strikingly, Rad51 IRIF was rescued in Brca1 Δ11/Δ11 , 53bp1 −/− cells, but foci remained relatively low in Brca1 CC/CC , 53bp1 −/− cells (Fig. 4E).The latter observations are in line with previous work showing that in the absence of 53bp1, Brca1 hypomorphic proteins that interact with Palb2 are required for efficient Rad51 loading and HR 35,36 .

RPA persists through mitosis with ART558
We next considered the consequences of DNA end resection proficiency on DNA repair outcomes during ART558 treatment.First, Western blotting measurements of γH2ax indicated that, in contrast to PARPi treatment where DNA damage was present in all cell lines, ART558 treatment only induced detectable increases in γH2ax in Brca1 CC/CC cells (Fig. 5A).Unrepaired replicative DNA damage often manifests as mitotic abnormalities and we measured the consequences of ART558 treatment on mitosis using H2B-mCherry labeling, which allows visualization and the live cell imaging of condensed chromosomes.Here, ART558 treatment marginally increased the number of abnormal mitoses in Brca1 +/+ and Brca1 Δ11/Δ11 cells.In contrast, the number of abnormal mitoses and lagging chromosomes, as well as micronuclei, dramatically increased in Brca1 CC/CC cells treated with ART558 (Fig. 5B and Supplementary Fig. 3b).These data suggest that ART558 minimally impacts the overall levels of spontaneous replication-associated breaks in Brca1 +/+ and Brca1 Δ11/Δ11 cells, but increases DNA damage in Brca1 CC/CC cells, leading to mitotic abnormalities.
We hypothesized that our inability to detect significant levels of DNA damage with ART558 treatment in asynchronous Brca1 Δ11/Δ11 cells might be due to transient damage that is repaired.To test this idea, we carried out imaging of synchronized cells post-thymidine release and monitored the presence and resolution of ssDNA overhangs and DNA breaks using RPA-mCherry and GFP-MDC1, respectively.Here, Brca1 Δ11/Δ11 cells that were MDC1 foci-positive post-thymidine release showed a trend for resolution during mitosis.In contrast, the relative decrease in MDC1 foci was lessened in ART558 treated Brca1 Δ11/Δ11 cells, with daughter cells demonstrating 1.9-fold more MDC1 foci per nucleus relative to vehicle-treated cells (Fig. 5C).RPA-mCherry foci were rarely detected by live cell imaging methods in Brca1 Δ11/Δ11 cells.
In Brca1 CC/CC cells, while most cells with MDC1 foci showed resolution during mitosis in vehicle-treated cells, ART558 treatment resulted in the persistence of foci through mitosis, with daughter cells demonstrating 2-fold more foci per nucleus (Fig. 5C).Furthermore, in vehicle-treated Brca1 CC/CC cells, most RPA foci resolved during mitosis.However, with ART558 treatment, the number of RPA foci were significantly elevated pre-mitosis and showed only partial resolution in mitosis, with daughter cells demonstrating 4.2-fold more foci per nucleus (Fig. 5C).We surmise that during ART558 treatment, Brca1 Δ11/Δ11 cells have limited resection of breaks that arise during replication, providing compatible NHEJ substrates, which are repaired prior to, or following mitosis.In contrast, Brca1 CC/CC cells frequently resect DSBs, generating suitable TMEJ, but poor NHEJ substrates 11 , and as a consequence, RPA-coated ssDNA overhangs and DNA damage persist with ART558 treatment.
DNA breaks that endure throughout the cell cycle can be aberrantly joined, potentially accounting for the increase in complex marker chromosomes and other clonal rearrangements observed in Brca1 mutant, Polq −/− cells (Fig. 2B).We sought to determine whether loss of Polθ activity directly increases the number of chromosome rearrangements in Brca1 CC/CC MEFs.To test this, we artificially induced DSBs in the Rosa26 and H3f3b mouse loci using CRISPR/Cas9 in Brca1 CC/CC cells that were treated with either DMSO or ART558 38 .Translocation frequency did not increase with ART558 (Fig. 5D, Supplementary Fig. 3c) and as expected, ART558 resulted in fewer junctions containing microhomology (Fig. 5E, Supplementary Fig. 3d).Interestingly, vehicle-treated cells had a median deletion size of 18.5 bp, but the median deletion in ART558 treated Brca1 CC/CC cells was 86 bp (Fig. 5E).Additionally, insertions were observed with ART558 treatment (Supplementary Fig. 3d).We speculate larger deletions induced by ART558 treatment could be due to resection associated single-strand annealing (SSA) repair, or cleavage of overhangs to generate NHEJ substrates.Thus, when DNA end resection is active, loss of Polθ activity results in persisting DNA breaks, with some chromosome fragments eventually aberrantly joined, perhaps by SSA or NHEJ.

Discussion
HR repair gene mutations often impart a dependence on alternative DSB repair pathways, providing therapeutic opportunities that selectively target cancer cells.Cancers with BRCA1 and BRCA2 mutations are thought to be highly dependent on Polθ activity for their survival 21,22 .Furthermore, the hyperactivation of DNA end resection, through the loss of proteins that inhibit this pathway, also induces Polθ-dependent survival 11,32,33 .Thus, our understanding of the interplay between HR gene mutations, loss of end resection factors, and susceptibility to Polθi is continuing to emerge.The presence of hypomorphic BRCA proteins, which lack certain domains and functions, while retaining others, adds complexity to existing paradigms.In the current work, we show that the underlying HR gene mutation, and its effect on DNA end resection, is a crucial determinant of cellular TMEJ addiction and the response to Polθi.Furthermore, that synthetic sickness may be a more appropriate description of the Brca1 and Polθ genetic interaction, given Polθ loss could be tolerated for viability in Brca1 mutated, 53bp1 wild-type cells.
Surprisingly, Brca1 Δ11/Δ11 , Polq −/− cells were readily generated, but grew more slowly.Moreover, Brca1 Δ11/Δ11 cells showed moderate ART558 sensitivity.The mouse Brca1 Δ11 allele expresses a truncated protein that lacks exon 11.Brca1 Δ11 cells and mice have been extensively characterized and shown to be defective in counteracting 53bp1 and promoting DNA end resection 1 .However, because microhomology can often be found within ssDNA overhangs that are less than 20 base pairs 41 , we speculate that a portion of DSBs have long enough overhangs to engage Polθ, accounting for the mild ART558 sensitivity observed in Brca1 Δ11/Δ11 cells.In human cancers, BRCA1 frameshift mutations located in exon 11 account for up to one-third of all pathogenic mutations (https://research.nhgri.nih.gov/projects/bic/index.shtml).Cancer cells with these mutations are also capable of expressing an exon 11 deficient isoform through alternative splicing and removal of exon 11, and this protein is similarly defective for promoting resection 26 .Furthermore, cells with mutations that cause a complete loss of BRCA1 protein expression are also defective for DNA end resection 30 .Thus, our data suggest that a significant portion of patients with BRCA1 mutation-containing cancers might show a limited response to Polθi monotherapy.
Brca1 CC/CC , Polq −/− cells had phenotypes that were more severe, with greater numbers of chromosome alterations, including numerous marker chromosomes.We developed the Brca1 CC allele to demonstrate the importance of the Brca1-Palb2 complex for Brca2-Rad51 loading, establishing that Brca1 has additional functions in HR beyond DNA end resection that cannot be readily bypassed 35 .However, BRCA1 CC domain missense mutations in patients with cancer are relatively uncommon.Nonetheless, the Brca1 CC allele is a useful tool to separate Brca1 functions in HR, and is phenotypically similar to Palb2 and Brca2deficient cells, with proficient DNA end resection, but severely disrupted Rad51 loading.Hence, we extended our findings to show that in human isogenic cells, PALB2-and BRCA2-deficient cells were more sensitive to ART558 than BRCA1-deficient cells.However, sensitivity was dependent on the presence of BRCA1, which was required to promote DNA end resection.The implications of these data are that cancers with PALB2 and BRCA2 inactivating mutations may be the optimal group for Polθi monotherapy.We acknowledge that some BRCA1 cancers are responsive to Polθi, although loss of end resection factors could contribute 32,33,42 .Furthermore, in future studies, it will be important to test additional Polθi such as novobiocin, as well as determine possible differences between pharmacological inhibition and genetic KO of Polθ.
The biological mechanisms by which Polθ enzymatic activities support the viability of HR-deficient cells are undergoing investigation, and include roles in replication fork gap filling 34,43,44 , and DSB repair 15 .
In our experiments, we did not detect elevated γH2ax in asynchronous Brca1 Δ11/Δ11 cells with ART558 treatment.We speculate that limited resection of DSBs provides compatible substrates for NHEJ, which repairs DNA damage in interphase, and circumvents the requirement for TMEJ-repair.TMEJ was recently shown to be active in mitosis 45,46 , which is supported by our results, indicating that DSBs remain present in cells undergoing mitosis during ART558 treatment.In end resection proficient cells, we observed elevated RPA32 in pre-mitotic cells that persisted to daughter cells with ART558, suggesting that TMEJ is also active prior to mitosis, where it may be required to strip RPA from ssDNA 47 .
Polq −/− cells were previously shown to have a reduced incidence of translocations between loci subject to CRISPR-based cutting 21 .In contrast, Brca1 CC/CC , Polq −/− cells harbored a plethora of rearranged chromosomes.We predict that in Brca1 CC/CC , Polq −/− cells, rearrangements accumulate due to failed repair of endogenous replication-associated DSBs, and accumulating chromosome fragments are eventually randomly joined as cells continue to proliferate.Differences could also be due to replication-associated single-ended DSBs versus CRISPR-induced two-ended DSBs.Brca1 CC/CC cells treated with ART558 also had larger deletions at translocation junction sites, potentially due to active resection, SSA, or nucleases that adapt ends for NHEJ.It remains to be seen whether patient tumors will demonstrate extensive chromosome interchanges post-Polθi treatment.We speculate implications of such Polθi-induced chromosomal alterations could include cGAS-STING activation, which could prime tumors for immunotherapy 48,49 .
Taken together, our study establishes cellular DNA end resection proficiency as a critical feature of the HR-Polθi synthetic lethal paradigm.Additionally, persisting ssDNA-coated RPA could be a source of Polθi-induced chromosomal rearrangements and cellular toxicity.The dissection of specific protein functions within HR revealed that Polθi monotherapy may provide greater benefit for cancers with PALB2 and BRCA2 mutations relative to those with BRCA1 mutations.Our data supports the study of RPA foci as a possible predictive biomarker for Polθi sensitivity in cancers with HR gene mutations.Clinical trials underway combine Polθi with PARPi or DNA-damaging agents; our work, along with others, supports this strategy, particularly for those patients carrying BRCA1 mutations.Moving forward, specific mutationto-function relationships should be considered in the application of DNA repair inhibitor therapeutics.

Mouse breeding and genotyping
All experiments involving animals were approved by the Fox Chase Cancer Center (FCCC) Institutional Animal Care and Use Committee and housed in standard conditions.The generation of the Brca1 CC mouse allele is previously described 35 .We obtained mice harboring Polq mutant, 53bp1 mutant from Jax labs, and Brca1 Δ11 alleles from the NCI.Mice were from mixed backgrounds.Genotyping of mice was performed with DNA extracted with 50 mM NaOH.Brca1 CC genotypes were determined by PCR amplification using the following primers: forward GAGGAGCTACAGTGAAGCAGATCT, reverse TATGA-CAAGGCACGTGTGGA.Subsequently, the 1.2 kb PCR product was subjected to an EcoNI restriction digest of DNA from wild-type mice showed 1 band, whereas DNA from heterozygous mice had 3 bands, and homozygous mutant had 2 bands.Brca1 Δ11 mice were genotyped using two PCR reactions that indicate the presence of wild-type (465 bp band) or the Δ11 allele (~650 bp band) using the following primers: common forward CTGGGTAGTTTGTAAGCATGC, wild-type reverse CAATAAACTGCTGGTCTCAGG, Δ11 reverse CTGCGAG-CAGTCTTCAGAAAG. 53bp1 mice were genotyped using two PCR reactions that indicate the presence of wild-type (~450 bp band) or the 53bp1 allele (~450 bp band) using the following primers: wild-type forward CTCCAGAGAGAACCCAGCAG, mutant forward CTAAAGCG-CATGCTCCAGAC, common reverse GAACTTGGCTCACACCCATT. Polq mice were genotyped using two PCR reactions, one to detect the wild-type allele (~200 bp band) and one to detect the mutant allele (~300 bp band) with the following primers: wild-type forward TGCAGTGTACAGATGTTACTTTT, wild-type reverse TGGAGGTAG-CATTTCTTCTC, mutant forward TCACTAGGTTGGGGTTCTC, mutant reverse CATCAGAAGCTGACTCTAGAG.

MEF generation, Polq knockout, and cell culture
MEFs were generated and transduced with SV40 large T antigen as previously described and maintained in Dulbecco's Modified Eagle's Medium with 4.5 g/L glucose, 15% fetal bovine serum, 1× GlutaMAX, 1× MEM nonessential amino acids, 1 mM sodium pyruvate, 1× penicillin/ streptomycin 35 .MEFs were tested for Mycoplasma using the Lonza MycoAlert Plus kit (Lonza, LT07-710) and genotypes were confirmed using the assays described above.

Growth curves and drug responses
Cell growth curve assays were performed for the indicated periods of time using the Sartorius Incucyte imaging system.Exponentially growing cells were seeded into 24-well plates at 1000 cells per well for MEFs.Four images were collected per well at each time point and confluence measurements averaged.A minimum of three biological replicates were performed and the mean confluence was presented with SEM for all replicates.Colony formation assays were performed in six-well plates with 300 MEF cells per well or 500 MDA-MB-436 cells per well in media with the indicated concentrations of ART558 or rucaparib.DMSO concentrations were equalized for each condition.Cells were fixed with 4:1 methanol:acetic acid after 2 weeks and then stained with crystal violet.Colonies were then counted and IC50 was calculated using results from three or more independent experiments.Response curves are shown as mean colony formation ± S.E.M.

Karyotypic and M-FISH Analysis
Preparation of mouse metaphases and G-banding were carried out as previously described 50 .Cells in logarithmic growth phase were treated with 0.03 μg/ml Colcemid, trypsinized, transferred to hypotonic 0.075 M KCl for 15 min at 37 degrees then fixed in 3:1 methanol: acetic acid.The guidelines used for karyotypic designations and chromosome breakpoint determination of murine metaphase chromosomes are located at http://www.pathology.washington.edu/research/cytopages/idiograms/mouse/.For mFISH analysis, metaphase preparations were hybridized using a 21Xmouse multicolor FISH probe for mouse chromosomes mFISH kit (MetaSystems, Heidelberg, Germany).The probe consists of 21 painting probes specific for the 21 different mouse chromosomes, labeled with different fluorochromes, and the excitation/emission spectra are comparable to aqua, green, orange, red, and near-infrared fluorochromes.Image capturing and processing were performed with a Zeiss AxioImager Z2 fluorescence microscope, with single bandpass filters (Chroma Technology) appropriate for each fluorochrome and an Isis/mFISH image analysis system (MetaSystems).

Digital droplet PCR reporter assays
HR and TMEJ were measured by ddPCR assay as previously described 51 .In brief, Cas9 and Rosa26 targeted sgRNA were electroporated then repair products were amplified across the Cas9-induced break site in the Rosa26 locus.HR was measured through the incorporation of a homologous repair template using forward primer CCGCCCA-TAGTACTCTGGAG and reverse AGAAAACTGGCCCTTGCCATT.The repair signal was normalized to 100 copies of genomic DNA using a control ddPCR reaction on chromosome 6.HR assay quantifications are presented as a percentage genomic DNA copies, determined by the chromosome 6 control reaction.TMEJ values were determined by summing the quantifications of three validated TMEJ repair products (del23bp, del39bp, del95bp) and normalizing them to the total TMEJ repair products obtained in wild-type cells.The primers for each TMEJ product are as follows: 23bp forward TTTAAGCCTGCCCAGATC, 39bp forward TTTAAGCCTGCCCAGATC, 95bp forward GCCCACACACC AGTCC, and reverse TCAGTTGGGCTGTTTTGGAG.

END-seq resection measurements
To measure DNA end resection by END-seq, cells were transduced with a retroviral vector encoding doxycycline-inducible expression of estrogen receptor fused nuclease AsiSI (provided by Andre Nussenzweig).Singlecell clones were isolated and selected based on their doxycyclineinduced expression levels and nuclear translocation by adding tamoxifen to the media.END-seq was performed as described previously 52 .In brief, exponentially growing cells were treated for 19 h with 4 mg/ml doxycycline to induce AsiSI expression, followed by the addition of 1 μM tamoxifen for 5 h to stimulate AsiSI nuclear localization.DNA ends were processed, biotinylated adapters ligated, and breaks isolated by streptavidin capture followed by PCR amplification and paired-end sequencing using an Illumina NextSeq 2000 and a P2 100 cycle flow cell.Reads were trimmed (Trimmomatic v0.36), PCR duplicates removed (Picard v2.27.5), and mapped to the mouse GRCm38/mm10 genome with BWA v0.7.17-r1188.Subsequent files were generated with SAMtools v1.13 and BEDtools v2.27.1.Normalized read counts were binned for the 20 kb upstream and downstream of each AsiSI genomic location, identified as 5'-GCGATCGC-3' sequences within the mouse genome.Cut AsiSI locations were determined based on peak detection and ordered by resection distance calculated from peak width.Binned read counts are plotted for each cut AsiSI location and shown as heatmaps, centered on the AsiSI genomic site.Resection quantifications are shown for the top 20 resected sites for each sample.END-seq data are available at the GEO repository under accession number GSE244865.

Live cell imaging
An H2B-mCherry construct was generated by cloning H2B from Addgene plasmid #113086 (gift of Janet Rossant) in an pENTR1A plasmid containing mCherry then transferred to the pLX304 expression plasmid using LR clonase (Invitrogen, 11791020) according to the manufacturer's instructions.MEFs were transduced with lentivirus containing the H2B-mCherry plasmid and selected with Blasticidin.Cells were seeded into Petri dishes with coverslip bottoms (Ibidi, 8128-200) and imaged with a Leica STELLARIS 5 confocal microscope with temperature and carbon dioxide regulation.Three fields of view were imaged per condition for n = 3 biological replicates.Maximum projection images were generated and a minimum of 10 mitotic cells were tracked manually per replicate.Abnormal versus normal mitoses were counted and the type of abnormality observed (lagging chromosomes, anaphase bridges, multipolar spindles) was quantified.
The GFP-MDC1 and RPA2-mCherry plasmids were constructed from Addgene plasmids #26427 (gift of Eric Campeau) and #102613 (gift of Marc Wold) and transduced into MEFs as described above.Cells were additionally sorted for dual expression of GFP and mCherry using a BD FACSAria II system.For GFP-MDC1 and RPA2-mCherry imaging, cells were preincubated with 2 mM thymidine to synchronize in S phase, released for 2 h to allow for replication restart, then treated with DMSO or 10 μM ART558, and imaged.Adjacent fields of view (×20) were collected and stitched together using a Leica STELLARIS 5 confocal microscope and software.Maximum intensity projections were generated and images of individual nuclei were generated for focipositive nuclei.A total of 15 foci containing nuclei across n = 2-3 replicates were quantified for the number of foci per nucleus over time.Foci were counted using an ImageJ (v.2.1.0/1.53c)macro 6 .Foci identification was performed using the ImageJ background subtraction, contrast, and Gaussian blur functions followed by thresholding to generate a foci mask image.The threshold values were applied consistently within each experiment and manually confirmed.The ImageJ analyze particles function was then used to quantify foci for each nucleus from the foci mask image.

Translocation analysis
Translocation assays were adapted from a previous study 21 .A single cell Brca1 CC/CC clone expressing doxycycline-inducible Cas9 was isolated and expanded following transduction with lentivirus containing pCW-Cas9 plasmid (Addgene plasmid #50661, gift from Eric Lander and David Sabatini).The following single guide RNA sequences targeting Rosa26 and H3f3b were cloned into pLenti-F2 and lentivirus generated: ACTCCAGTCTTTCTAGAAGA and GTTGGCTCGCCGGA-TACGGG.For transduction with sgRNA plasmid, 500 cells per well were plated in 96 well plates and the following day were transduced with virus, 1× polybrene (Santa Cruz Biotechnology, sc-134220), and 4 μg/ml doxycycline in the presence of DMSO or 10 μM ART558.Negative controls were generated by excluding sgRNA lentivirus.DNA was collected 96 h after transduction by replacing media with 60 μl of 50 mM NaOH and heated to 95 degrees for 30 min followed by the addition of 15 μl 1 M Tris-HCl pH 8.0 from 5 wells per condition.Cutting at both loci was assessed using the following primers and Sanger sequencing: RosaF GCGGGAGAAATGGATATGAA, RosaR GCACGTT TCCGACTTGAGTT, H3f3bF TTGACGCCTTCCTTCTTCTG, H3f3bR AAC CTTTGAAAAAGCCCACA.
Amplification of a Rosa-H3f3B translocation product was performed by PCR using the H3f3bF and RosaR primers by a nested PCR reaction using the following primers: H3f3bNF CTGCCATTCCAGA-GATTGGT and Rosa26NR TCCCAAAGTCGCTCTGAGTT.The first round of PCR was performed with 500 ng genomic DNA for each well and 4 μl product for the nested reaction, each using 60 degrees annealing temperature.The nested PCR reactions were run on an agarose gel with expected translocation junctions of ~500 bp.Wells containing amplified translocation products were counted and product size was estimated.Technical replicates were performed for each well using 500 ng genomic DNA inputs.Translocation frequency was determined from the total number of translocation-positive wells across both technical replicates and shown for each n = 4 biological replicates.For breakpoint sequence analysis, PCR products from individual wells were sequenced by Sanger sequencing and deconvolved using DECODR v3.0 software (decodr.org) 53.If multiple Sanger sequence traces were detected and could not be reliably deconvolved, those sequences were excluded from further analyses.Of note, ART558 treatment resulted in frequent deletions of varying sizes visible on the agarose gels leading to fewer sequences available for breakpoint analyses.Sequences were aligned to the expected translocation junction and assessed for microhomology, insertions, and deletions at each nucleotide position along the expected product.Data are presented as microhomology or deletion frequencies at each position, centered at the breakpoint.Additionally, total deletion sizes were determined for each sequence and shown separately.

qRT-PCR analysis
RNA was extracted from cell pellets following manufacturer instructions (Qiagen, 74104).Reverse transcription with M-MLV (Thermo Fisher, 28025013) and PCR using the PowerUp SYBR Green Master Mix (Thermo Fisher, A25743) were performed using conditions recommended by the manufacturer and the following primers: Polq-F CTGTATGCCTCTGGCTTTCTC, Polq-R CTTCAGCTGCTTCCTCTTCTT.Polq expression was normalized to 36b4 (Rplp0) expression, determined using the following primers: F GCTCCAAGCAGATGCAGCA, R CCGGATGTGAGGCAGCAG.

Statistics and reproducibility
Statistical tests, number of replicates, and p values are described in the figure legends.GraphPad Prism Version 9.4.0 was used for unpaired, two-tailed t tests and comparisons indicated in the figures.R and RStudio v1.3.1093 were used for chi-square goodness of fit tests comparing expected and observed genotypes.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Fig. 1 |
Fig. 1 | Assessment of cellular and organismal Polq −/− phenotypes.A Cartoon showing (left), Brca1 alleles and protein products with domain functions indicated; (Right), Brca1 functions in HR (simplified), the Brca1-Δ11 protein is defective for DNA end resection; the Brca1-CC protein is defective for Palb2-Brca2-Rad51 loading.B Western blotting for the indicated proteins in MEF cell lines.C Cartoon showing (left), process for generating CRISPR/Cas9 manipulated MEFs.(Right), Polθ protein domains and predicted effect of stop codon from sgRNA targeted exon 4 of Polq.D Polq gene mutations were binned according to whether frameshift mutations in both alleles (−/−) (green), or retaining at least one allele with a missense or one frameshift mutation (+/+, +/−) (blue).See Supplementary Data 1.E Mice with the indicated genotypes were intercrossed, and expected and observed genotypes were shown.A representative photograph of mice with the indicated genotypes is shown.See Supplementary Fig. 1e.P values are obtained from two-sided chi-square goodness of fit tests for the binomial of each genotype.F Mice with the indicated genotypes were intercrossed, and expected and observed genotypes were shown.P values were obtained from chi-square goodness of fit tests comparing expected and observed genotypes.Source data are provided as a Source Data file.

Fig. 3 |
Fig. 3 | ART558 and PARPi sensitivity profiles.A Cells were incubated with increasing concentrations of rucaparib for 2 weeks and colony formation assessed.Mean and S.E.M. colony formation as well as mean IC50 values, n = 4 biological replicates.Fold changes in IC50 relative to Brca1 +/+ , 53bp1 +/+ (WT) are shown.B Cells were incubated with increasing concentrations of ART558 for 2 weeks and colony formation assessed, as for A. Source data are provided as a Source Data file.

Fig. 4 |
Fig. 4 | Genotype-conferred DNA repair profiles.A HR repair was measured by ddPCR following Cas9-induced DSB generation in cells of the indicated genotypes.HR measurements are quantifications of repair products generated from a homologous template as a proportion of the non-templated sequence and presented as a percentage of genomic DNA copies determined by a control reaction for n = 3 biological replicates.Mean and S.E.M. are shown.B TMEJ was measured by ddPCR for 3 distinct repair products, summed, and normalized to the TMEJ products detected in wild-type MEFs for n = 3 biological replicates.Mean and S.E.M. are shown.C END-seq was used to quantify resection for MEFs expressing the nuclease AsiSI with the indicated genotypes.(Top) Heatmaps of normalized, binned END-seq signal centered at AsiSI sites and extending 20 kb in both directions.(Bottom) Resection distances were calculated for each AsiSI site and quantifications shown by Tukey box and whisker plot for the top 20 resected sites for each sample, with the box drawn from the 1st to 3rd quartile and median indicated with a line.D Cells were (2 Gy) γ-irradiated and after 6 h pre-extracted, fixed, and assessed for Rpa foci formation using immunofluorescence staining.Mean and S.E.M. percentage of cells with more than 5 foci for n = 3 biological replicates.E Cells were assessed for Rad51 foci as described in D. F Brca1 Δ11/Δ11 , 53bp1 −/− cells were treated with siRNA targeting the Δ11 isoform of Brca1 (B1) or scrambled control siRNA (Sc).Left, western blotting confirmed knockdown of the Brca1-Δ11 isoform.Right, Cells were assessed for Rad51 foci formation as in D. Mean and S.E.M. percentage of cells with more than five foci for n = 3 biological replicates.G Cells from F were seeded in the presence of DMSO or 1 μM ART558 and the indicated siRNA and colony formation were measured.Colony counts were normalized to DMSO-treated controls for independent experiments and mean and S.E.M were shown for n = 3 biological replicates.H Model demonstrating the interplay between 53bp1 and Brca1 mutations.Statistical significance was assessed by unpaired, two-tailed t tests.Source data are provided as a Source Data file.

Fig. 5 |
Fig. 5 | DNA damage and translocations with ART558.A Brca1 +/+ , Brca1 CC/CC , and Brca1 Δ11/Δ11 MEFs were treated with DMSO, ART558, or rucaparib for 24 h, and whole cell extracts were collected to assess γH2ax by western blotting.Representative blots from three independent experiments are shown.B MEFs expressing H2B-mCherry were tracked after 72 h of DMSO or 10 μM ART558 treatments.(Top) The percentage of nuclei with micronuclei present are shown for individual replicates (bar is the median).(Bottom) Individual mitoses were classified as normal or abnormal with mean and S.E.M. percentages for n = 3 biological replicates.Representative images and examples of lagging chromosomes (orange arrow) and anaphase bridges (pink arrow) are shown.Scale bar is equal to 10 μm.See Supplementary Fig. 3b for additional images.C Brca1 CC/CC and Brca1 Δ11/Δ11 MEFs were treated with thymidine for 16 h and then released into fresh media.DMSO or 10 μM ART558 was added 2 h after release and live cells were imaged for GFP-MDC1 and RPA2-mCherry.MDC1 and RPA2 foci were quantified for 15 individual cells that entered mitosis with foci present over three independent experiments.The mean and S.E.M. number of foci are shown prior to mitosis (P), during mitosis (M), and in daughter cells (D).Representative images are shown.D Translocation frequency in Brca1 CC/CC cells was determined after inducing breaks in Rosa26 and H3f3b loci.Cells growing in 96 well plates with DMSO or 10 μM ART558 were compared to wells with no break induction (−).The average percentage of wells with detectable translocation products is shown for n = 4 biological replicates (bar is median).See Supplementary Fig. 3c.E Breakpoint junctions for individual wells from D were sequenced and aligned to the predicted translocation product.(Left) Deletion size was determined for each sequence.(Right) The frequency of deletions (green) and microhomologies (orange) are shown at each coordinate of the predicted translocation product.If multiple sequence traces were detected and could not be deconvolved, those sequences were excluded from analyses, see Supplementary Fig. 3d.Statistical significance was assessed for the indicated comparisons by unpaired, two-tailed t tests.Source data are provided as a Source Data file.

Fig. 6 |
Fig. 6 | HR deficiencies and ART558 sensitivity.A MDA-MB-436 clones with control sgRNA targeting BFP, or PALB2 knockout with and without ectopic BRCA1 addback.Expression of the indicated proteins was assessed by western blotting.B RPA foci formation was measured in geminin-positive cells 8 h after 10 Gy γirradiation.Cells with >10 RPA foci were counted as positive.Mean and S.E.M. shown for n = 3 biological replicates.C As for B, cells with 5 or more RAD51 foci were counted positive.D Cells were seeded into increasing concentrations of rucaparib and colony formation was measured after 2 weeks.Mean and S.E.M from n = 3 biological replicates.E Cells were seeded into increasing concentrations of ART558 and colony formation was measured after 2 weeks.Mean and S.E.M from n = 3 biological replicates.See Supplementary Fig. 4. F Model depicts TMEJ usage in BRCA1 proficient and deficient settings.We propose that BRCA1 deficiency leads to moderate TMEJ dependence due to minimal DNA end resection.In contrast, BRCA1 +/+ cells are proficient in DNA end resection resulting in hyper-dependence on TMEJ in the absence of RAD51 loading caused by PALB2 or BRCA2 mutations.Source data are provided as a Source Data file.